The present invention relates in general to the use of transgenic animals to produce therapeutically useful amounts of clinically important recombinant proteins. More particularly, this invention relates to the production in transgenic animals of clinically useful quantities of the blood clotting protein, fibrinogen (“FIB”).
The ultimate event in the blood clotting cascade is the thrombin-catalyzed conversion of FIB (Mr=340,000) to fibrin (Mr=329,000), the latter forming the fibrin clot. FIB deficiency is generally transmitted as an autosomal recessive trait and may manifest as a complete or partial absence of FIB from the blood plasma. Clinically, the disease resembles moderate or mild hemophilia. Congenital fibrinogen abnormality may be due to the hereditary synthesis of structurally or functionally abnormal molecules, as in Vlissingen, Ijmuiden and Nijmegen fibrinogens. An acquired deficiency of this protein may occur due to impaired hepatic synthesis of the protein, as occurs, for example, in hepatitis or hepatic necrosis, or to accelerated destruction of the protein caused, for example, by increased blood proteolytic activity.
Control of bleeding in such patients is currently achieved by transfusion of FIB contained in freshly-frozen human plasma or in concentrates of the protein isolated from donor blood. While these replacement therapies are generally effective, they place patients at risk for virus-transmissible diseases such as hepatitis or AIDS. Although this risk has been greatly reduced by inactivating such viruses with heat or organic solvents, such preparations have greatly increased the cost of treatment, and are not risk free. There is thus a critical need for a source of this protein alternate to human plasma.
An important advance in obtaining an alternate clinical source of FIB has been the cloning of cDNAs encoding the three different fibrinogen chains, and the publication of cDNA sequences. Rixon et al., Biochemistry 22: 3237 (1983); Chung et al., ibid: 3244; Chung et al., ibid: 3250. The structure of the FIB molecule is exceedingly complex. Each molecule of FIB consists of two sets of three different polypeptide chains, designated Aα, Bβ and Gγ, with molecular masses of 66 kDa, 52 kDa and 46.5 kDa, respectively. The two half-molecules containing each set of chains are linked together by three disulfide bonds. In addition, a complex set of intra- and inter-chain disulfide bonds (there are a total of 29 disulfide bonds with no free sulfhydryl groups) are involved in maintaining proper functional structure. Further, FIB is a glycoprotein with highly specific glycosylations. The molecule contains four carbohydrate chains, one each on the B, β, G and γ chains; the α and A chains contain no carbohydrate. About 11 kDa of the total molecular mass of FIB (340 kDa) is attributable to this carbohydrate, added to the molecule post-translationally. In addition, isoforms of glycoproteins are known corresponding to differences in sialic acids on the carbohydrate chains. Proper carbohydrate modification is required for functional activity of FIB.
These highly complex characteristics of the functional FIB molecule has made unpredictable and difficult the expression, assembly and secretion of fully formed and functional recombinant molecules. A cDNA encoding the human FIB Aα chain has been expressed in bacteria. Lord, DNA 4:33 (1985). This is of limited usefulness, however, since the other fibrinogen chains that bear carbohydrates cannot be produced in prokaryotes.
Individual FIB chains have been expressed in COS1 (transformed monkey kidney fibroblast) cells. Danishevsky et al., Biochim. Biophys. Acta 1048: 202 (1990). In addition, transfecting COS1 cells with a combination of cDNAs encoding the individual human fibrinogen subunit chains is reported to produce the holoprotein, but the amounts produced were small, and substantially less than the production achieved in the transgenic animal systems to be described below. Roy et al., J. Biol. Chem., 266: 4758 (1991). The secretion of partially assembled or wholly unassembled and separate human FIB (“hFIB”) or recombinant human FIB (“rhFIB”) chains has not been reported for native or genetically engineered tissues. Chung et al. (1983); Danishevsky et al (1990). In addition, there are serious drawbacks to the use of mammalian cell tissue culture systems for production of FIB. These include the high costs of growth media, the labor intensive nature of such systems, and limited production capacity.
An important need persists for an efficient and relatively inexpensive means of producing clinically useful amounts of infectious particle-free rhFIB protein. The present invention satisfies this need. It has been surprisingly found that transgenic animals can be genetically engineered to produce and secrete into readily accessible body fluids therapeutically useful quantities of rhFIB. In addition to therapeutic uses involving replacement or addition therapy, the FIB of the invention finds us in a variety of applications, such as a “glue” in surgical procedures, as a delivery system for drugs, such as antibiotics or anti-parasitic agents, to wounds, as a food substitute, and for altering the composition of milk. These transgenic systems are described below.